1. Field of the Invention
The present invention generally relates to a process for the production of human recombinant polypeptides using transformed mammalian cells. More particularly, the present invention relates to a process for producing enhanced quantities of biologically active recombinant protein using mammalian cells cultured in horizontally rotating culture vessels.
2. Description of the Related Art
In vitro cultures of animal cells are hosts for an increasing assortment of recombinant protein products. Recombinant proteins derived from animal cells are employed in a range of disciplines from agriculture to medicine to basic research. Progress made in genetic engineering and animal cell cultivation has enabled the production of an increasing assortment of recombinant proteins in sufficient quantities for the development of new drug therapies (e.g., plasminogen activator).
At present, both prokaryotes and eukaryotes are utilized as hosts for commercial production of recombinant proteins. The choice of one over the other is based on the structural complexity of the protein being produced, the desired yield and cost effectiveness. In cases where the native protein is not post-translationally modified, or where post-translational modification does not significantly affect protein function, the recombinant protein can be produced in prokaryotic organisms such as bacteria. Prokaryotes are preferred because their doubling times are in hours, rather than the days required for animal cell doubling. Likewise, prokaryotes yield grams of protein per liter of media, rather than the milligrams per liter produced in animal cells. Problems with the production of quantities of biologically active recombinant proteins for commercial use have encouraged investigators to use other types of cells, such as insect cells to try to increase the quantity of produced protein.
Yet there are many important human proteins that must undergo extensive post-translational processing and modification after syntheses of the amino acid chain itself (e.g., glycosylation, phosphorylation or macromolecular assembly) to be functional. Bacteria do not have the mechanisms for the correct post-translational modification of human proteins. While insect and non-human mammalian cells and transgenic animals can produce modifications similar to human cells, there are clear differences. For instance a recent study comparing glycosylation of recombinant human gamma interferon in Chinese hamster ovary (“CHO”) cells, Sf9 insect cells and the mammary gland of transgenic mice showed that in each case, the patterns of glycosylation were distinctly different from that of the naturally occurring protein [James, D. C. et al. 1995]. Such differences can influence the secretion, biological activity, antigenicity and stability of the protein. For example, current techniques used to produce complex recombinant proteins such as Epogen, produced by Amgen, do not provide for the proper posttranslational modification of the protein. The degree of this problem is illustrated in an article in Science 291:2338, 2001 stating that Amgen had to discard 80% of its recombinant Epogen because of incorrect glycosylation.
Because animal cells are not enclosed in a cell wall like bacteria, they are susceptible to hydrodynamic forces within a bioreactor. Agitation, shear and other hydrodynamic phenomena have a profound effect on cell morphology and physiology, which can result in cell damage and death [Spaulding et al. 1997]. From a morphological perspective, hydrodynamic forces alter cell shape, adhesion, and membrane integrity, which can lead to cell death from lysis, detachment of anchorage-dependent cells from surfaces, and reduced metabolic activity [Croughan, M. et al. 1989 and Petersen, J. F. et al. 1988].
There are two main types of bioreactors that have been used in recombinant protein production in mammalian cells (i.e., the stirred tank and the perfused hollow fiber bioreactor). In perfused, hollow fiber bioreactors, cells are grown on one side of a semi-permeable membrane while the medium is perfused on the other side. Oxygen and nutrients can diffuse from the media compartment to the cell compartment. Metabolic wastes and secreted products can diffuse from the cell compartment to the media compartment. The advantages of this system include the lack of mechanical stress applied and the easy recovery of secreted proteins. However, the cells are grown on the surface of the fibers and the fibers limit cell growth to two dimensions. In addition, because the secreted proteins diffuse into the media compartment the secreted proteins are diluted in a large volume of media making downstream purification more difficult and expensive. Furthermore, the hollow fiber bioreactors are limited in size and are not practical to scale-up to large volumes.
The stirred-tank reactor is the system of choice for most companies because of its flexibility. These reactors can maintain cells in suspension with or without microcarriers by agitation through mechanical stirring with an impeller or gas bubble sparging. The stirred-tank reactors can be operated in different feed modes, and can be scaled up to very large volumes (e.g., 10,000 liters) These systems provide a large surface area for cell growth and the efficient transfer of nutrients, oxygen and metabolic wastes. The stirred-tank reactor maintains a homogenous environment throughout the reactor, and prevents cells from settling by a continuous stirring or mixing of the components within the reactor. But in the stirred-tank bioreactors, mixing can also cause cell damage from the resultant hydrodynamic forces attributed to bulk-fluid turbulence and gas/liquid interfaces [Kunas, K. T. et al. 1990]. Typically, these interfaces arise during cultivation as a result of sparging, vortex formation, turbulent eddies, fluid-wall shear gradients and surface oxygenation. Thus, a major disadvantage of the stirred-tank reactor is that shear stress causes significant cell injury and death, leading to lower levels of recombinant protein production, or lower yield.
In each of these conventional bioreactors, some recombinant proteins cannot be produced in sufficient quantities or a high enough specific activity to allow the proteins to be used for commercial purposes. This is particularly a problem in the production of human proteins of medical interest. It is speculated that the production of such proteins in human cells could provide a protein with greater specific activity, or higher bioreactivity, thereby reducing the quantity of protein needed to give a therapeutic result.
There is a continuing need to improve mammalian, particularly human, cell culture techniques to achieve commercial scale recombinant protein production. In addition, there is a continuing need to develop techniques providing appropriately post-translationally modified proteins having sufficient bioactivity for therapeutic purposes. Furthermore, a need exists for a means of producing the recombinant protein in a high enough concentration to ease the time and expense of subsequently purifying the protein.